The present invention relates to the field of molecular interactions, and particularly to methods and devices related to the controlled binding and dissociation of the members of a pair of chemical entities having a pH-dependent affinity.
An affinity pair is a pair of two molecules that mutually bind with high specificity. For many affinity pairs, mutual binding occurs through formation of multiple non-covalent bonds such as hydrogen bonds, electrostatic bonds, polarization interactions, Van der Waals forces and hydrophobic interactions. Although any one individual such non-covalent bond is relatively weak, the sum of the contributions of many such non-covalent bonds is such that the attraction (the affinity) between the two molecules making up the affinity pair is very strong. The high specificity of binding of affinity pairs is often a result of the fact that affinity is a function of the sum of many non-covalent interactions, the strength of which is for the most part highly geometry-dependent. For this reason, affinity pair association is often described using a “lock and key” or “puzzle piece” analogy.
The members of affinity pairs are known to belong to different and varied groups of molecules. One common type of affinity pair is an antibody/antigen pair.
An antibody is a protein produced by an organism that binds to an antigen, a molecule that is frequently, but not always, a peptide or protein. Antibody to antigen bonding is highly specific, so that a given antibody generally binds only to a specific antigen through a part of the antigen called an epitope.
As noted above, binding between the members of an affinity pair is through non-covalent bonds, including electrostatic attraction, polarization interactions and hydrogen bonds between protonable functional groups. Further, many types of molecules that are members of an affinity pair, such as peptides and proteins, have a conformation that is in a large measure determined by non-covalent interactions such as electrostatic interactions and internal hydrogen bonds between protonatable groups (e.g., secondary and/or tertiary structure). The degree of protonation of a molecule is in a large part determined by the availability of protons in the environment (most often an aqueous solution, but also others such as gels and the like) in which the molecule is found. In such cases, the affinity pair is characterized as having a pH-dependent affinity. The pH of the environment in which the affinity pair is found influences the degree to which the affinity pair is bound or dissociated by determining the affinity of the affinity pair by influencing the conformation of one or both of the members of the affinity pair and by influencing the degree of protonation of the functional groups through which the members of the affinity pair bind.
The pH dependence of the affinity of an affinity pair and consequence pH dependence on the degree of binding of an affinity pair has been used, for example, for affinity chromatography in the isolation of antibodies from a solution. A specific antigen is immobilized on a matrix to make a stationary phase. An eluent including the desired antibody, that together with the immobilized antigen constitutes an affinity pair, is passed along the stationary phase when the pH of the solution is such that the affinity of the affinity pair is high. Due to the high affinity and the high specificity of the affinity pair, only the antibody binds to the stationary phase. The stationary phase is washed with a washing phase having a pH where the affinity of the affinity pair is high, washing away undesired molecules while the antibody remains bound to the stationary phase. Subsequently, the bound antibodies are released from the stationary phase with an elution solution having a pH where the affinity of the affinity pair is low. The affinity pair dissociates and the antibody is isolated. A disadvantage of affinity chromatography is the requirement to provide and use different solutions having different pHs.
It would be useful to accurately control the degree of binding or dissociation of affinity pairs having a pH-dependent affinity without the use of different solutions having different pHs.